Fractionation of protein containing mixtures

ABSTRACT

Thus, a primary aspect of the present invention relates to a method for the fractionation of a protein-containing mixture wherein the protein-containing mixture is selected from the group consisting of milk, milk derived products, milk derived raw materials, vegetable derived products, vegetable derived extracts, fruit derived products, fruit derived extracts, fish derived products, and fish derived extracts, the method comprising the steps of: a) optionally adjusting the pH of the mixture; b) applying the mixture to an adsorption column comprising an adsorbent, the adsorbent comprises a particle with at least one high density non-porous core, surrounded by a porous material, the adsorbent having a particle density of at least 1.5 g/ml and a mean particle size of at most 150 μm; c) optionally washing the column; d) eluting at least one protein from the adsorbent.

FIELD OF THE INVENTION

The invention relates to an industrial production process and method forthe isolation and fractionation of biomolecular substances, particularlyproteins in milk and whey products as well as other milk derived rawmaterials. The selection of adsorbent allows for industrial scaleseparation of proteins from large volumes of liquids.

BACKGROUND OF THE INVENTION

Milk is one of the most thoroughly researched foods in history.Countless scientific papers document milk's composition and describe thebiological functionalities in this complex bio-resource. Proteins,peptides, enzymes and other biomolecular substances constitute a majorand very important fraction in milk and are believed responsible formany of the specific functionalities passed on from a mother to hernew-born in addition to basic nutrients.

During the past two decades, there has been significant focus onutilisation of bovine whey proteins. Today, several bovine Whey ProteinConcentrates (WPC) and bovine Whey Protein Isolates (WPI) are standardproducts obtained through various membrane filtration techniques as wellas ion exchange adsorption procedures. Further utilisation of the bovinewhey in terms of fractionation of the proteins into individual proteinfractions, such as β-lactoglobulin, α-lactalbumin, immunoglobulins,lactoperoxidase, and lactoferrin, is made possible throughchromatographic packed bed separation techniques. Protein products fromchromatographic separation technologies are generally characterised bytheir low- to non-fat content and are useful for a broad range ofapplications e.g. within food, feed, functional foods, and health careproducts.

Since the first market introductions of WPC and WPI products and morerecently the first purified single protein products (e.g. lactoferrinand lactoperoxidase) there has evolved an ever increasing demand foreven more sophisticated and still more efficient and cost effectiveproductions methods.

Among the various industrial chromatographic separation techniquesdeveloped in recent years, Expanded Bed Adsorption (EBA) has beensuccessfully introduced to the certain fields of biotechnology industry.EBA is a type of fluidised bed adsorption wherein the level ofback-mixing is kept at a minimum. Compared with other chromatographicseparation technologies, EBA offer a significant advantage because itcan be used directly with non-clarified feed.

During EBA, the adsorbent bed is allowed to expand inside the columnwhen a flow of liquid is applied (see FIG. 1). Expansion/fluidisation ofthe bed is often effected in a column having provided at each of itsends a net structure covering the cross-sectional area of the column, orsome other perforated devices, which will not generate turbulence in theflow. See, for instance, WO-A-9218237 (Amersham Pharmacia Biotech AB,Sweden). The same effect has also been observed in a system utilising astirred inlet flow WO-A-9200799, (UpFront Chromatography A/S). Inaddition, other distributors are likely to be feasible.

In the expanded bed state, the distance between the adsorbent particlesresult in a free passage of particulate impurities in the feed stream.By contrast, traditional packed beds work as depth filters that canclog, resulting in increased back-pressure unless the feed is thoroughlyclarified. Since no significant pressure builds up in an EBA column, itis possible to apply EBA without the limitations in size and flow ratenormally associated with packed-bed columns.

An EBA process is characterised by very limited back-mixing of theliquid inside the column as opposed to the well know turbulent fluidisedbeds typically employed for chemical reactions. Back-mixing in a bed isoften measured as axial dispersion (“vessel dispersion number”), seeLevenspiel, “Chemical Reaction Engineering” 2nd Edition, John Wiley &Sons (1972).

The adsorbent media employed in an EBA process must have a higherdensity than the feed stock in order to produce acceptable flow ratesduring operation. If the density is too low, the media will be lost inthe column effluent. Generally, EBA adsorbent particles may either bedesigned to be impermeable to the fluid, in which case the availablesurface area per unit volume is small; or particles may be designed tobe permeable to the fluid, in which case the material chosen has to havethe correct density per se. Unfortunately, the most interestingmaterials for many applications, e.g. materials such as natural andsynthetic polysaccharides like agar, alginates, carrageenans, agarose,dextran, modified starches, and celluloses; synthetic organic polymersand copolymers typically based on acrylic monomers used forchromatographic purification of proteins in packed bed columns are notof suitable density per se. Therefore, these materials are not readilyapplied in EBA.

However, certain types of organic polymers and certain types of silicabased materials may be produced to provide carrier particles of suitabledensity, but such carriers may not at the same time be suitableadsorbents, e.g. for protein purification procedures, where suchmaterials may provide low permeability, non-specific interactions anddenature bound proteins. Further, for such polymers, it may be difficultand expensive to design derivatisation schemes for affinitychromatography media. In addition, certain types of permeable silicaparticles have been used for EBA. However, the properties of thesematerials are far from optimal. Thus, the materials are unstable at pHabove 7, fragile to shear forces, and provide non-specific interactions.In addition solid silicate materials have a maximal density of approx.2.5 g/mL.

The density of the adsorbent media may be controlled by an inert,high-density core incorporated in the polymer phase (composite media,conglomerates see e.g. WO-A-9200799). High-density core materials aretypically chosen from high density materials such as glass, quartz orheavy metals either in the form of an alloy such as stainless steel oran oxide (e.g. zirconium oxide) or some other metal salt (e.g. tungstencarbide). The core material may also comprise metal spheres (e.g.tantalum). The core material of the particles may vary in size andshape. Typical sizes are within 5-80 micrometers.

In EBA, as a result of the optimisation of the characteristics of theadsorbent media (size distribution), plug-flow conditions with verylittle back-mixing is obtained inside the column. The plug-flowbehaviour is crucial in order to obtain an efficient adsorption.

Today, several important bio-pharmaceuticals are being produced usingthe EBA technology. However, no commercial processes for milk and wheyfractionation are based on EBA so far. This is in great part due to thelarge scale of the process required for milk and whey fractionation,typically involving extremely high volumes of raw material to be treatedper day (e.g. several m³/hour) which requires extremely high efficiencyand productivity of the EBA system. Current processes are not capable,in practice, to achieve the level of performance for these and certainother raw materials.

A major supplier of EBA adsorbents and EBA columns is UpFrontChromatography A/S, Denmark. These products are supplied under thetrademark FastLine (see e.g.WO 92/00799, UpFront Chromatography A/S,Denmark), which discloses a large number of fillers and polymericmaterials that can be combined to produce composite beads,conglomerates) intended for adsorption in EBA.

Amersham Pharmacia Biotech AB, Sweden markets StreamLine which utiliseporous beads of agarose with quartz particles as filler material(WO-A-9218237, Pharmacia Biotech AB).

Another supplier is Bioprocessing Ltd. (Durham, England) whose porousglass beads (Prosep0) can be used for chromatography on expanded beds(Beyzavi et al, Genetic Engineering News, Mar. 1, 1994 17).

WO 97/17132 (Amersham Pharmacia Biotech) discloses a population of beadshaving a density >1 g/cm³ and comprising a polymer base matrix in whicha particulate filler is incorporated. The beads are characterized inthat the filler particles have a density >3 g/cm³ and in that thedensity and/or size of the beads are distributed within predetermineddensity and size ranges. Particularly important filler particles arethose which have rounded shapes, for instance spheres, ellipsoids oraggregates/agglomerates thereof. The bead population is particularlyusable in adsorption processes in fluidized beds, with preference tostable expanded beds.

WO 00/57982 discloses a particulate material having a density of atleast 2.5 g/mL, where the particles of the particulate material have anaverage diameter of 5-75 μm, and the particles of the particulatematerial are essentially constructed of a polymeric base matrix, e.g. apolysaccharide such as agarose, and a non-porous core material, e.g.steel and titanium, said core material having a density of at least 3.0g/mL, said polymeric base matrix including pendant groups which arepositively charged at pH 4.0 or which are affinity ligands for abio-molecule. Possible pendant groups include polyethyleneimine (PEI),diethylaminoethyl (DEAE) and quaternary aminoethyl (QAE). The materialsare useful in expanded bed or fluidised bed chromatography processes, inparticular for purification of bio-macromolecules such as plasmid DNA,chromosomal DNA, RNA, viral DNA, bacteria and viruses.

WO-A-8603136 (Graves and Burns; University Patents Inc) discloses beadscontaining magnetic filler particles and their use in fluidized bedsstabilized by an externally applied magnetic field. See also Burns etal., Biotechnol. Bioengin. 27 (1985) 137-145.

WO-A1-9833572 (Amersham Pharmacia Biotech) discloses a method foradsorption of a substance from a liquid sample on a fluidized bead orstirred suspension, in which the beads used comprise a base matrix andexhibit a structure having affinity to the substance, characterized inthat the structure is covalently bound to the base matrix via anextender. Populations of beads in which the beads contain a fillerincorporated in a base matrix and an extender are also described.

In chromatography on packed beds it has earlier been suggested to useporous beads, the pores of which wholly or partly have been filled withhydrophilic gels carrying affinity ligands, such as ion exchange groups.One example is Macrosob-K which is macroporous kieselguhr which has beenfilled with agarose which in turn has been derivatized to exhibit DEAEor CM ion exchange groups (Macrosorb-KAX.DEAE and Macrosorb KAX.CM,respectively (GB-A-1,586,364, Miles). This latter type of materials havealso been applied in fluidized bed chromatography (Bite et al., In:Verrall et al., Separations for Biotechnology (1987), Ellis Horwood LTD,Chapter 13, 193-199).

U.S. Pat. No. 4,976,865 (Sanchez, et al, CNRS) teaches fluidised bedsand the use of segmented columns to mimic the multi-step adsorptiontaking place in packed as well as stabilised expanded beds for isolationof whey compounds. The beads used in the experimental part are silicaparticles (Spherosil, density=1.4 g/mL, mean particle size=225 μm) thathave been coated. The linear flow rate implemented in the experimentalpart is 1.3×10⁻³ m/s, which is equal to 468 cm/hour). The experimentalparts discloses the use of this type of fluidized bed adsorption forseparation of biological macromolecules from whey. There is nodisclosure of any flow rates and/or binding capacities obtainable withadsorbents having a lower than 225 μm mean particle size.

Immunoglobulins—or antibodies—constitute a very important class ofproteins which are present in various body fluids of mammals, birds andfish functioning as protective agents of the animal against substances,bacteria and virus challenging the animal. Immunoglobulins are typicallypresent in animal blood, milk, and saliva as well as other body fluidsand secretions.

All the above mentioned applications of immunoglobulins requires somesort of isolation of the antibody from the crude raw material, but eachkind of application has its own very varying demands with respect to thefinal purity and allowable cost of the antibody product.

Generally, there exists a very broad range of different methodsavailable for isolation of immunoglobulins giving a very broad range offinal purities, yields and cost of the product.

Traditional methods for isolation of immunoglobulins are based onselective reversible precipitation of the protein fraction comprisingthe immunoglobulins while leaving other groups of proteins in solution.Typical precipitation agents being ethanol, polyethylene glycol,lyotropic (anti-chaotropic) salts such as ammonium sulfate and potassiumphosphate, and caprylic acid.

Typically, these precipitation methods are giving very impure productswhile at the same time being time consuming and laborious. Furthermore,the addition of the precipitating agent to the raw material makes itdifficult to use the supernatant for other purposes and creates adisposal problem. This is particularly relevant in relation to the largescale purification of immunoglobulins from for instance, whey.

Ion exchange chromatography is another well known method of proteinfractionation frequently used for isolation of immunoglobulins. However,this method is not generally applicable because of the restraints inionic strength and pH necessary to ensure efficient binding of theantibody together with the varying isoelectric points of differentimmunoglobulins.

Protein A and Protein G affinity chromatography are very popular andwidespread methods for isolation and purification of immunoglobulins,particularly for isolation of monoclonal antibodies, mainly due to theease of use and the high purity obtained. Although being popular it ishowever recognised that Protein A and Protein G poses several problemsto the user among which are: very high cost, variable binding efficiencyof different monoclonal antibodies (particularly mouse IgG₁), leakage ofProtein A/Protein G into the product, and low stability of the matrix intypical cleaning solutions, e.g. 1 M sodium hydroxide. Each of thesedrawbacks have its specific consequence in the individual application,ranging from insignificant to very serious and prohibitive consequences.

Hydrophobic chromatography is also a method widely described forisolation of immunoglobulins, e.g. in “Application Note 210, BioProcessMedia” published by Pharmacia LKB Biotechnology, 1991. In thispublication, a state of the art product “Phenyl Sepharose HighPerformance” is described for the purpose of purifying monoclonalantibodies from cell culture supernatants. As with other hydrophobicmatrices employed so far it is necessary to add lyotropic salts to theraw material to make the immunoglobulin bind efficiently. The boundantibody is released from the matrix by lowering the concentration oflyotropic salt in a continuous or stepwise gradient. It is recommendedto combine the hydrophobic chromatography with a further step if highlypure product is the object.

The disadvantage of this procedure is the necessity to add lyotropicsalt to the raw material as this gives a disposal problem and therebyincreased cost to the large scale user. The addition of lyotropic saltsto the raw materials would in many instances be prohibitive in largescale applications as the salt would prevent any economically feasibleuse of the immunoglobulin depleted raw material in combination with theproblem of disposing several thousand liters of waste.

Thiophilic adsorption chromatography was introduced by J. Porath in 1985(J. Porath et al; FEBS Letters, vol.185, p.306, 1985) as a newchromatographic adsorption principle for isolation of immunoglobulins.Porath describes the technology wherein divinyl sulfone-activatedagarose in combination with various ligands comprising a freemercapto-group demonstrate specific binding of immunoglobulins in thepresence of 0.5 M potassium sulfate, i.e. a lyotropic salt. It waspostulated that the sulfone group, from the vinyl sulfone spacer, andthe resulting thio-ether in the ligand was a structural necessity toobtain the described specificity and capacity for binding of antibodies.It was, however, later shown that the thio-ether could be replaced bynitrogen or oxygen if the ligand further comprised an aromatic radical(K. L. Knudsen et al, Analytical Biochemistry, vol 201, p.170, 1992).

Although the matrices described for thiophilic chromatography generallyshow good performance, they also have a major disadvantage in that it isneeded to add lyotropic salts to the raw material to ensure efficientbinding of the immunoglobulin, which is a problem for the reasonsdiscussed above.

Other thiophilic ligands coupled to epoxy activated agarose have beendisclosed in (I. Porath et.al., Makromol. Chem., Makromol. Symp., vol.17, p.359, 1988) and (A. Schwarz et.al., Journal of Chromatography B,vol. 664, pp. 83-88, 1995), e.g. 2-mercaptopyridine,2-mercaptopyrimidine, and 2-mercaptothiazoline. However, all theseaffinity matrices still have inadequate affinity constants to ensure anefficient binding of the antibody without added lyotropic salts.

To avoid the above mentioned problems and disadvantages theinvestigators of the present invention have developed a method for largescale fractionation, purification and isolation of at least one proteinfrom a protein-containing mixture. This method is applicable forindustrial use, it can handle very large volumes of a protein-containingmixture, it is fast and it provides a highly purified protein.

The investigators of the present invention also found that it waspossible to carry out the fractionation, purification and isolation ofproteins without the use of lyotropic salts.

SUMMARY OF THE INVENTION

Accordingly a general aspect of the present invention relates to amethod of fractionating large volumes of protein-containing solutionsusing adsorbent which, despite having a small particle diameter and avery high density, exhibit a high binding capacity for the desiredprotein.

One object of the present invention is to provide a process forindustrial-scale fractionation of proteins from raw materials using EBAmethodology by selection of EBA adsorbents. As stated, this object isachieved at least in part by providing an EBA process utilising aspecific adsorbent selected according to its particle diameter andparticle density. This allows an EBA process for operating efficientlyat linear flow rates above at least 200 cm/hour.

Thus, a primary aspect of the present invention relates to a method forthe fractionation of a protein-containing mixture wherein theprotein-containing mixture is selected from the group consisting ofmilk, milk derived products, milk derived raw materials, vegetablederived products, vegetable derived extracts, fruit derived products,fruit derived extracts, fish derived products, and fish derivedextracts, said method comprising the steps of: a) optionally adjustingthe pH of the mixture; b) applying said mixture to an adsorption columncomprising an adsorbent, said adsorbent comprises a particle with atleast one high density non-porous core, surrounded by a porous material,the adsorbent having a particle density of at least 1.5 g/ml and a meanparticle size of at most 150 μm; c) optionally washing the column; d)eluting at least one protein from the adsorbent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows an illustration of the expansion of the adsorbent bed byan upward flow of liquid, compared to a traditional packed bed.

FIG. 2: Shows a flow sheet diagram of an EBA process according to theinvention.

FIG. 3: Shows a modular whey Protein fractionation facility, capturinglactoferrin and lactoperoxidase in Step I, and bovine serum albumin,p-lactoglobulin and immunoglobulin in Step II.

FIG. 4: Shows a microscope picture of stainless steel/agarose EBAadsorbent particles.

FIG. 5: Shows a SDS-PAGE illustrating the purity of lactoferrin andlactoperoxidase. Lane 1 illustrates the whey, lane 2 illustrates thewash, lane 3 illustrates the LP-eluate and lane 4 illustrates theLF-eluate.

FIG. 6: Shows a SDS-PAGE illustrating the elution profiles of wheyproteins as a function of binding pH. Lane 1 illustrates the molecularmarker, lane 2 illustrates eluate 1 at pH 4, lane 3 illustrates eluate 2at pH 4, lane 4 illustrates eluate 1 at pH 4.5, lane 5 illustrateseluate 2 at pH 4.5, lane 6 illustrates eluate 1 at pH 5, lane 7illustrates eluate 2 at pH 5, lane 8 illustrates the molecular weightmarker, lane 9 illustrates eluate 1 at pH 5.5, lane 10 illustrateseluate 2 at pH 5.5, lane 11 illustrates eluate 1 at pH 6 and lane 12illustrates eluate 2 at pH 6.

FIG. 7: Shows a SDS-PAGE showing purity of BSA/β-LG and IgG fractions.Lane 1 illustrates whey (LF and LP depleted), lane 2 illustrates thewash with 2.5 mg/ml caprylic acid pH 6 and lane 3 illustrates theeluate, eluted with 20 mM NaOH (170 ml).

FIG. 8: Shows a SDS-PAGE showing the results obtained by the sequentialwashing/elution of β-LG and BSA and the final elution of IgG. Lane 1illustrates wash with 50 mM sodium acetate pH 5.5, lane 2 illustratesthe wash with 2.5 mg/ml caprylic acid pH 6 and lane 3 illustrates theeluate, eluted with 20 mM NaOH.

FIG. 9: Shows microscope picture of spherical UpFront tungstencarbide-8-agarose particles. The mean particle size is 59 μm and thedensity is 3.3 g/ml.

FIG. 10: Shows the expansion curve for three different tungsten carbide8-agarose particles. A) density 2.4 g/ml and mean particle size 56 μm,B) density 3.3 g/ml and mean particle size 59 μm and C) density 3.2 g/mland mean particle size 90 μm.

FIG. 11: Shows the particle distribution of tungsten carbide 8-agaroseparticles having a density of 2.4 g/ml and a mean particle size of 56 μmand 552 particles was measured.

FIG. 12: Shows the particle distribution of tungsten carbide 8-agaroseparticles having a density of 3.3 g/ml and a mean particle size of 59 μmand 602 particles was measured.

FIG. 13: Shows the particle distribution of tungsten carbide 8-agaroseparticles having a density of 3.2 g/ml and a mean particle size of 90 μmand 283 particles was measured.

FIG. 14: Shows a microscopic picture a spherical UpFront tungstencarbide-25-agarose particles. The mean particle size is 77 μm and thedensity is 3.7 g/ml.

FIG. 15: Shows the expansion curve on UpFront tungstencarbide-25-agarose having a mean particle size is 77 μm and a density is3.7 g/ml.

FIG. 16: Shows the particle distribution of UpFront tungstencarbide-25-agarose particles having a density of 3.7 g/ml and a meanparticle size of 77 μm and 332 particles was measured.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly the present invention provides a method for fractionation ofa protein-containing mixture by utilisation of an equilibrated adsorbentcolumn containing adsorbents comprising small sized particles having alarge density. This selection of size and density allows an increasedflow rate and makes it feasible to handle large volumes for industrialscale fractionation of protein-containing mixtures and obtaining highlypure proteins.

Protein-Containing Mixture

The method according to the present invention is targeted for industrialor large-scale fractionation processes where large volumes must behandled. Suitably, the protein-containing mixture is selected from thegroup comprising of milk, and milk derived products such as whey andother milk derived raw materials, vegetable derived products andextracts, fruit derived products and extracts, fish derived products andextracts. Typically, the protein-containing mixture is a milk-derivedproduct, preferably selected from milk and whey.

In the present context, the term “protein-containing mixture” relates toa mixture of biological origin which comprises at least one protein orbiomolecular substance needed to be fractionated, partially or whollypurified or isolated on a industrial or large scale. Typical mixturesinclude milk, skim milk, whey and other milk derived raw materials,vegetable derived products and extracts, fruit derived products andextracts, fish derived products and extracts.

The degree to which fractionation can be utilized in any particulardairy depends on the existing exploitation of the whey. If a productionof WPC is running, it is possible to extract minor protein fractions andstill sustain adequate WPC for an existing market. It may even bepossible to extract one or more of the major whey proteins at low yieldwithout harming an existing WPC market. In situations with norestrictions, it is possible to obtain multiple protein products fromthe whey stream.

pH Adjustment

In the dairy industry the process integration of EBA for whey proteinfractionation is typically made in a cheese-making facility immediatelyfollowing the removal of fines and whey cream. The whey may need anadjustment in pH (depending on the protein of interest), the ligandchemistry, and the type of whey.

In a preferred embodiment of the present invention theprotein-containing mixture is pH adjustment to a pH facilitating thecapture of the protein or proteins to be isolated, prior to beingapplied to the adsorbent column. This pH may be adjusted to a pH valueselected in the entire pH range, preferably from pH 2-13, morepreferably from pH 3-11.

Adsorbent Column

The adsorbent column to be used may be any kind suitable for either EBA(Expanded Bed Adsorption) or suitable for packed bed adsorption or acombination thereof. The adsorbent column may be used in either a batchsystem or in a continues system. In the present context the term“adsorbent column” relates to any kind of container which can besupplied with at least one inlet and at least one outlet for theapplication of the mixture to the column and subsequent to elute theprotein.

The fact that the EBA technology generally can work efficiently withnon-clarified raw materials makes it attractive to implement for theisolation and fractionation of biomolecular substances from milk andwhey. Compared to processes based on packed bed adsorption techniquesEBA may offer a robust process comprising fewer steps and thus result inincreased yields and an improved process economy. Due to the expansionof the adsorbent bed during execution of an EBA process, EBA columns mayfurther be scaled up to industrial scale without any significantconsiderations regarding increased back pressures or breakdown of theprocess due to clogging of the system which often is a problem whenusing packed bed columns. However, the present state of art within theEBA technology does not adequately address the specific problemsassociated with the treatment of extremely high volumes of rawmaterials, such as but not limited to milk and whey.

It is a further object of this invention to provide a process forisolation and fractionation of biomolecular substances from milk, skimmilk, whey and other milk derived raw materials based on adsorption toany type of solid phase material of any shape and format includingpacked bed adsorption, batch adsorption, suspended bed adsorption, EBAand membrane based adsorption characterised by the use of selectiveligand chemistry enabling the specific binding and subsequent elution ofsubstantially only one biomolecular substance, or alternatively enablinga group specific binding of a few biomolecular substances followed byselective and consecutive elution of one or more substances from theadsorbent.

General Expansion Bed Adsorption technology is known to the personskilled in the art and the method of the present invention may beadapted to the processes described in, for example, WO 92/00799, WO92/18237, WO 97/17132, WO 00/57982 and WO 98/33572.

Adsorbent

As stated, it is an overall object of the present invention to provide anovel process for isolation and fractionation of biomolecular substancesfrom whole milk, skim milk, whey and other milk derived raw materials,which process is based on the use of EBA methodology and EBA adsorbentsfulfilling the needs for implementing efficient EBA processes forisolation and fractionation of such biomolecular substances in anindustrial scale. An initial but optional step in the process of theinvention typically involves the equilibration of the adsorbent. In apreferred embodiment, the equilibration liquid is water such as tapwater, demineralised water, water produced by reverse osmosis ordistilled water.

In the present context the term “adsorbent” relates to the entire bedpresent in the adsorbent column and the term “adsorbent particle” areused interchangeably with the term “particle” and relates to theindividual single particles which makes up the adsorbent.

The flow rate, the size of the particles and the density of theparticles all have influence on the expansion of the fluid bed and it isimportant to control the degree of expansion in such a way to keep theparticles inside the column. The degree of expansion may be determinedas H/H0, where H0 is the height of the bed in packed bed mode and H isthe height of the bed in expanded mode. In a preferred embodiment of thepresent invention the degree of expansion H/H0 is in the range of1.0-20, such as 1.0-10, e.g. 1.0-6, such as 1.2-5, e.g. 1.5-4 such as4-6, such as 3-5, e.g. 3-4 such as 4-6. In an other preferred embodimentof the present invention the degree of expansion H/H0 is at least 1.0,such as at least 1.5, e.g. at least 2, such as at least 2.5, e.g. atleast 3, such as at least 3.5, e.g. at least 4, such as at least 4.5,e.g. at least 5, such as at least 5.5, e.g. at least 6, such as at least10, e.g. at least 20.

The density of the EBA adsorbent particle is found to be highlysignificant for the applicable flow rates in relation to the maximaldegree of expansion of the adsorbent bed possible inside a typical EBAcolumn (e.g. H/H0 max 3-5) and must be at least 1.3 g/mL, morepreferably at least 1.5 g/mL, still more preferably at least 1.8 g/mL,even more preferably at least 2.0 g/mL, most preferably at least 2.3g/mL in order to enable a high productivity of the process.

In a preferred embodiment of the present invention the adsorbentparticle has a mean particle size of at most 150 μm, particularly atmost 120 μm, more particularly at most 100 μm, even more particularly atmost 90 μm, even more particularly at most 80 μm, even more particularlyat most 70 μm. Typically the adsorbent particle has a mean particle sizein the range of 40-150 μm, such as 40-120 μm, e.g. 40-100, such as40-75, e.g. 40-50 μm.

In a combination of preferred embodiments, where the average particlediameter is 120 μm or less, the particle density is at least 1.6 g/mL,more preferably at least 1.9 g/mL. When the average particle diameter isless than 90 μm the density must be at least 1.8 g/mL or more preferableat least 2.0 g/mL. When the average particle diameter is less than 75 μmthe density must be at least 2.0 g/mL, more preferable at least 2.3 g/mLand most preferable at least 2.5 g/mL.

The high density of the adsorbent particle is, to a great extent,achieved by inclusion of a certain proportion of a dense non-porous corematerials, preferably having a density of at least 4.0 g/mL, such as atleast 5.0, Typically, the non-porous core material has a density in therange of about 4.0-25 g/ml, such as about 4.0-20 g/ml, e.g. about 4.0-15g/mL, such as 12-19 g/ml, e.g. 14-18 g/ml, such as about 6.0-15.0 g/mL,e.g. about 6.0-10 g/ml.

Subsequently, the protein-containing mixture is loaded and thebiomolecular substance(s) of interest are adsorbed, typically underpressure. Particulate material and soluble impurities are optionallyremoved from the column during the wash.

The fractionation may thus be performed efficiently by applying theprotein-containing mixture to the adsorbent column at a linear flowrates of at least 3 cm/min, such as at least 5 cm/min, e.g. at least 8cm/min, such as at least 10 cm/min e.g. 20 cm/min. Typically the flowrate is selected in the range of 5-50 cm/min, such as in the range of5-15 cm/min, e.g. in the range of 10-30 cm/min, such as in the range of25-50 cm/min. These increased flow rates are possible to a great extentdue to the small particle size of the adsorbent.

Thus, in particular with regards to embodiments wherein the mixture ismilk or milk derived material such as milk or whey, the application rawmixture to the adsorbent column is with a linear flow rate of at least200 cm/hour, such as at least 300 cm/hour, more preferably at least 400cm/hour, such as at least 500 or 600 cm/hour, such as at least 900cm/hour.

When the protein-containing mixture is added to the adsorbent column theratio between the adsorbent particle present in the column and theprotein-containing mixture may be optimized in order to retain a highcapacity of the adsorbent column and to obtain a high purity of theprotein or proteins to be isolated. In a preferred embodiment of thepresent invention the adsorbent present in the column relative to theprotein-containing mixture to be loaded on to the column are provided ata ratio of at least 1:1000, such as at least 1:800, e.g. at least 1:600,such as at least 1:400, e.g. at least 1:300, such as at least 1:200,e.g. at least 1:100, such as at least 1:50, e.g. at least 1:30, such asat least 1:15, e.g. 1:10, such as 1:5 measured on a volume/volume basis.

Several parameters have an influence on the flow rate that can beimplemented in an EBA process. The fluidisation properties of theadsorbent particles (which may be described by the aid of Stokes Law)determine which flow rates that may be applied in order to expand theadsorbent and still keep it inside the column. The main factorsinfluencing this are the diameter and the density of the adsorbentparticles in combination with the viscosity of the liquid flowingthrough the column. However, the binding and mass transfer kineticsrelevant to a specific application are equally important to ensureoptimal efficiency and productivity of the EBA process. For example, itmay be possible to run an EBA column containing a certain EBA adsorbentat very high flow rates in terms of the physical fluidisation andexpansion properties, while the applied high flow rate results in a poorand inefficient adsorption (i.e. a low dynamic capacity) due to the factthat the target molecules to be bound cannot diffuse in and out of theadsorbent particles to match this flow rate (i.e. the mass transferkinetics is the limiting factor).

Consequently, in a combination of particularly preferred embodiments ofthe invention, where the applied linear flow rate during application ofthe raw material is above 300 cm/hour, the mean particle diameter isbelow 150 μm. Typically, in embodiments where the fractionation processis performed at an applied linear flow rate of above 500 cm/min, themean particle diameter is below 120 μm, preferably below 90 μm.Typically, in embodiments where the fractionation process is performedat an applied linear flow rate of above 600 cm/hour, the mean particlediameter is preferably below 85 μm, more preferably below 75 μm.

In a preferred embodiment of the present invention the adsorbentparticle has a density of at least 1.5 g/ml, such as at least 1.8 g/ml,e.g. at least 2.0 g/ml, such as at least 2.5 g/ml, such as at least 2.6g/ml, e.g. at least 3.0 g/ml, such as at least 3.5 g/ml, e.g. at least4.0 g/ml, such as at least 5 g/ml, e.g. at least 7 g/ml, such as atleast 10 g/ml, e.g. at least 15 g/ml.

The density of an adsorbent particle is meant to describe the density ofthe adsorbent in its fully solvated (e.g. hydrated) state as opposed tothe density of a dried adsorbent.

The adsorbent particle used according to the invention must be at leastpartly permeable to the biomolecular substance to be isolated in orderto ensure a significant binding capacity in contrast to impermeableparticles that can only bind the target molecule on its surfaceresulting in relatively low binding capacity. The adsorbent particle maybe of an array of different structures, compositions and shapes.

Thus, the adsorbent particles may be constituted by a number ofchemically derivatised porous materials having the necessary density andbinding capacity to operate at the given flow rates per se. Theparticles are either of the conglomerate type, as described in WO92/00799, having at least two non-porous cores surrounded by a porousmaterial, or of the pellicular type having a single non-porous coresurrounded by a porous material.

In the present context the term “conglomerate type” relates to aparticle of a particulate material, which comprises beads of corematerial of different types and sizes, held together by the polymericbase matrix, e.g. an core particle consisting of two or more highdensity particles held together by surrounding agarose (polymeric basematrix).

In the present context the term “pellicular type” relates to a compositeof particles, wherein each particle consists of only one high densitycore material coated with a layer of the porous polymeric base matrix,e.g. a high density stainless steel bead coated with agarose.

Accordingly the term “at least one high density non-porous core” relatesto either a pellicular core, comprising a single high density non-porousparticle or it relates to a conglomerate core comprising more that onehigh density non-porous particle.

The adsorbent particle, as stated, comprises a high density non-porouscore with a porous material surrounding the core, and said porousmaterial optionally comprising a ligand at its outer surface.

In the present context the term “core” relates to the non-porous coreparticle or core particles present inside the adsorbent particle. Thecore particle or core particles may be incidental distributed within theporous material and is not limited to be located in the centre of theadsorbent particle.

The non-porous core constitutes typically of at most 50% of the totalvolume of the adsorbent particle, such as at most 40%, preferably atmost 30%.

Examples of suitable non-porous core materials are inorganic compounds,metals, heavy metals, elementary non-metals, metal oxides, non metaloxides, metal salts and metal alloys, etc. as long as the densitycriteria above are fulfilled. Examples of such core materials are metalsilicates metal borosilicates; ceramics including titanium diboride,titanium carbide, zirconium diboride, zirconium carbide, tungstencarbide, silicon carbide, aluminum nitride, silicon nitride, titaniumnitride, yttrium oxide, silicon metal powder, and molybdenum disilide;metal oxides and sulfides, including magnesium, aluminum, titanium,vanadium, chromium, zirconium, hafnium, manganese, iron, cobalt, nickel,copper and silver oxide; non-metal oxides; metal salts, including bariumsulfate; metallic elements, including tungsten, zirconium, titanium,hafnium, vanadium, chromium, manganese, iron, cobalt, nickel, indium,copper, silver, gold, palladium, platinum, ruthenium, osmium, rhodiumand iridium, and alloys of metallic elements, such as alloys formedbetween said metallic elements, e.g. stainless steel; crystalline andamorphous forms of carbon, including graphite, carbon black andcharcoal. Preferred non-porous core materials are tungsten carbamide,tungsten, steel and titanium beads such as stainless steel beads.

The porous material is a polymeric base matrix used as a means forcovering and keeping multiple (or a single) core materials together andas a means for binding the adsorbing ligand.

The polymeric base matrix may be sought among certain types of naturalor synthetic organic polymers, typically selected from i) natural andsynthetic polysaccharides and other carbohydrate based polymers,including agar, alginate, carrageenan, guar gum, gum arabic, gum ghatti,gum tragacanth, karaya gum, locust bean gum, xanthan gum, agaroses,celluloses, pectins, mucins, dextrans, starches, heparins, chitosans,hydroxy starches, hydroxypropyl starches, carboxymethyl starches,hydroxyethyl celluloses, hydroxypropyl celluloses, and carboxymethylcelluloses; ii) synthetic organic polymers and monomers resulting inpolymers, including acrylic polymers, polyamides, polyimides,polyesters, polyethers, polymeric vinyl compounds, polyalkenes, andsubstituted derivatives thereof, as well as copolymers comprising morethan one such polymer functionally, and substituted derivatives thereof;and iii) mixture thereof.

A preferred group of polymeric base matrices are polysaccharides such asagarose.

From a productivity point of view it is important that the adsorbent isable to bind a high amount of the biomolecular substance per volume unitof the adsorbent. Thus we have found that it is preferable to applyadsorbents having a polymeric phase (i.e. the permeable backbone wherethe ligand is positioned and whereto the actual adsorption is takingplace) which constitutes at least 50% of the adsorbent particle volume,preferably at least 70%, more preferably at least 80% and mostpreferably at least 90% of the volume of the adsorbent particles.

The investigators of the present invention have found that in order toensure an efficient adsorption at high flow rates it is necessary tominimise the mean particle diameter of the adsorbent particle. Thus, ina preferred embodiment of the present invention the adsorbent particlehas a particle size of at the most 150 μm, typically a particle size inthe range of about 40 μm to 150 μm. The adsorbent particle typically hasa mean particle size of at most 120 μm, particularly at most 100 μm,more preferably at most 90 μm, 80 μm or 75 μm most preferably about 70μm.

The particles size analysis performed and referred to throughout thedescription and the examples is based on an computerised image analysisof the bead population giving the number of particles at any givenparticle diameter in relation to the total number of particles analysedin the specific measurement. Typically the total number of particlesanalysed will be in the range of 250-500 particles). These particle sizedata may be transferred into the volume percent represented by eachparticle size by a routine mathematical transformation of the data,calculating the volume of each bead and relating this to the totalvolume occupied by all beads counted in the measurement.

The particle size distribution according to the invention is preferablydefined so that more than 90% of the particles are found between 20-500%of the mean particle diameter, more preferable between 50-200% of themean particle diameter, most preferable between 50-150% of the meanparticle diameter.

The preferred shape of a single adsorbent particle is substantiallyspherical. The overall shape of the particles is, however, normally notextremely critical, thus, the particles can have other types of roundedshapes, e.g. ellipsoid, droplet and bean forms. However, for certainapplications (e.g. when the particles are used in a fluidised bedset-up), it is preferred that at least 95% of the particles aresubstantially spherical.

Preparation of the particulate material according to the invention maybe performed by various methods known per se (e.g. by conventionalprocesses known for the person skilled in the art, see e.g. EP 0 538 350B1 or WO 97/17132. For example, by block polymerisation of monomers;suspension polymerisation of monomers; block or suspension gelation ofgel-forming materials, e.g. by heating and cooling (e.g. of agarose) orby addition of gelation “catalysts” (e.g. adding a suitable metal ion toalginates or carrageenans); block or suspension cross-linking ofsuitable soluble materials (e.g. cross linking of dextrans, celluloses,or starches or gelatines, or other organic polymers with e.g.epichlorohydrin or divinyl sulphone); formation of silica polymers byacidification of silica solutions (e.g. block or suspension solutions);mixed procedures e.g. polymerisation and gelation; spraying procedures;and fluid bed coating of density controlling particles; coolingemulsions of density controlling particles suspended in polymeric basematrices in heated oil solvents; or by suspending density controllingparticles and active substance in a suitable monomer or copolymersolution followed by polymerisation.

In a particularly suitable embodiment generally applicable for thepreparation of the particulate material according to the invention, aparticulate material comprising agarose as the polymeric base matrix andsteel beads as the core material is obtained by heating a mixture ofagarose in water (to about 95° C.), adding the steel beads to themixture and transferring the mixture to a hot oil (e.g. vegetable oils),emulsifying the mixture by vigorous stirring (optionally by adding aconventional emulsifier) and cooling the mixture. It will be appreciatedby the person skilled in the art that the particle size (i.e. the amountof polymeric base matrix (here: agarose) which is incorporated in eachparticle can be adjusted by varying the speed of the mixer and thecooling process. Typically, following the primary production of aparticle preparation the particle size distribution may be furtherdefined by sieving and/or fluid bed elutriation.

The porous matrix, such as polymer agarose, is typically chemicallyderivatised with a low molecular weight compound referred to herein asthe ligand and the adsorbent comprise a ligand with affinity toproteins. The ligand constitutes the adsorbing functionality of theadsorbent media or the polymeric backbone of the adsorbent particle hasa binding functionality incorporated per se. Well-known ligandchemistries such as cation exchangers, e.g. sulphonic acid, have beenproven to be efficient tools for purification of whey proteins such aslactoferrin and lactoperoxidase. These proteins are positively charged,even at neutral pH, and selective interaction with a cation exchangercan be obtained. Other proteins require more sophisticated bindinginteraction with the ligand in order to obtain a selective adsorption.

Such affinity ligands, like the chargeable moieties, may be linked tothe base matrix by methods known to the person skilled in the art, e.g.as described in “Immobilized Affinity Ligand Techniques” by Hermanson etal., Academic Press, Inc., San Diego, 1992. In cases where the polymericbase matrix do not have the properties to function as an activesubstance, the polymeric base matrix (or matrices where a mixture ofpolymers are used) may be derivatised to function as an activesubstances in the procedures of activation or derivatisation. Thus,materials comprising hydroxyl, amino, amide, carboxyl or thiol groupsmay be activated or derivatised using various activating chemicals, e.g.chemicals such as cyanogen bromide, divinyl sulfone, epichlorohydrin,bisepoxyranes, dibromopropanol, glutaric dialdehyde, carbodiimides,anhydrides, hydrazines, periodates, benzoquinones, triazines, tosylates,tresylates, and diazonium ions.

Specifically preferred methods for chemical derivatization and specificligands applicable according to this invention is described in WO98/08603.

In order to ensure an optimal adsorption strength and productivity ofthe adsorbent it has been found that the ligand concentration on theadsorbent is very significant. Thus, in a suitable embodiment, theadsorbent carries ligands for adsorption of the biomolecular substancesin a concentration of at least 20 nM, such as at least 30 mM or at least40 mM, preferably at least 50 mM and most preferably at least 60 mM.

A subset of adsorbents may be characterised in terms of their bindingcapacity to bovine serum albumin (BSA). This subset of adsorbents aretypically those comprising a ligand selected from the group consistingof i) ligands comprising aromatic or heteroaromatic groups (radicals) ofthe following types as functional groups: benzoic acids such as2-aminobenzoic acids, 3-aminobenzoic acids, 4-aminobenzoic acids,2-mercaptobenzoic acids, 4-amino-2-chlorobenzoic acid,2-amino-5-chlorobenzoic acid, 2-amino-4-chlorobenzoic acid,4-aminosalicylic acids, 5-aminosalicylic acids, 3,4-diaminobenzoicacids, 3,5-diaminobenzoic acid, 5-aminoisophthalic acid, 4-aminophthalicacid; cinnamic acids such as hydroxy-cinnamic acids; nicotinic acidssuch as 2-mercaptonicotinic acids; naphthoic acids such as2-hydroxy-1-naphthoic acid; quinolines such as 2-mercaptoquinoline;tetrazolacetic acids such as 5-mercapto-1-tetrazolacetic acid;thiadiazols such as 2-mercapto-5-methyl-1,3,4-thiadiazol; benzimidazolssuch as 2-amino-benzimidazol, 2-mercaptobenzimidazol, and2-mercapto-5-nitrobenzimidazol; benzothiazols such as2-aminobenzothiazol, 2-amino-6-nitrobenzothiazol, 2-mercaptobenzothiazoland 2-mercapto-6-ethoxybenzothiazol; benzoxazols such as2-mercaptobenzoxazol; thiophenols such as thiophenol and2-aminothiophenol; 2-(4-aminophenylthio)acetic acid; aromatic orheteroaromatic sulfonic acids and phosphonic acids, such as1-amino-2-naphthol-4-sulfonic acid and phenols such as2-amino-4-nitro-phenol. It should be noted that the case where M isagarose, SP1 is derived from vinyl sulfone, and L is 4-aminobenzoic acidis specifically disclaimed in relation to the solid phase matricesaccording to the invention, cf. WO 92/16292, most preferablyamino-benzoic acids like 2-amino-benzoic acid, 2-mercapto-benzoic acid,3-aminobenzoic acid, 4-aminobenzoic acid, 4-amino-2-chlorobenzoic acid,2-amino-5-chlorobenzoic acid, 2-amino-4-chlorobenzoic acid,4-aminosalicylic acids, 5-aminosalicylic acids, 3,4-diaminobenzoicacids, 3,5-diaminobenzoic acid, 5-5-aminoisophthalic acid,4-aminophthalic acid; ii) ligands comprising 2-hydroxy-cinnamic acids,3-hydroxy-cinnamic acid and 4-hydroxy-cinnamic acid iii) ligandscomprising a carboxylic acid and an amino group as substituents such as2-amino-nicotinic acid, 2-mercapto-nicotinic acid, 6-amino-nicotinicacid and 2-amino-4-hydroxypyrimidine-carboxylic acid iv) ligandcomprising radicals derived from a benzene ring fused with aheteroaromatic ring system, e.g. a ligand selected from benzimidazolessuch as 2-mercapto-benzimidazol and 2-mercapto-5-nitro-benzimidazol;benzothiazols such as 2-amino-6-nitrobenzothiazol,2-mercaptobenzothiazol and 2-mercapto-6-ethoxybenzothiazol; benzoxazolssuch as 2-mercaptobenzoxazol;and v) ligands chosen from the group ofthiophenols such as thiophenol and 2-aminothiophenol.

Within the embodiment wherein the ligand is selected from group i)-v),the adsorbents typically have a dynamic binding capacity of at least 10g of biomolecular substance per liter, more preferably at least 20 g perliter, still more preferable at least 30 g per liter when testedaccording to the process conditions used in the relevant application.The binding capacity of the adsorbent may be determined in terms of itsbinding capacity to bovine serum albumin (BSA). The binding capacity istypically such that at least 10 g/L of BSA binds according to testMethod A.

Method A is a method used for determination of the bovine albuminbinding capacity of selected adsorbents consisting of the followingprocess:

Bovine serum albumin solution pH 4.0 (BSA pH 4.0): Purified bovine serumalbumin (A 7906, Sigma, USA) is dissolved to a final concentration of 2mg/ml in 20 mM sodium citrate pH 4.0. Adsorbents are washed with 50volumes of 20 mM sodium citrate pH 4.0 and drained on a suction filter.

A sample of 1.0 ml suction drained adsorbent is placed in a 50 ml testtube followed by the addition of 30 ml of BSA, pH 4.0.

The test tube is then closed with a stopper and the suspension incubatedon a roller mixer for 2 hours at room temperature (20-25° C.). The testtube is then centrifuged for 5 min. at 2000 RPM in order to sediment theadsorbent completely. The supernatant is then isolated from theadsorbent by pipetting into a separate test tube, avoiding thecarry-over of any adsorbent particles and filtered through a smallnon-adsorbing 0.2 μm filtre (Millipore, USA). Following this adetermination of the concentration of non-bound BSA in the supernatantis performed by measuring the optical density (OD) at 280 nm on aspectrophotometer.

The amount of BSA bound to the adsorbent is then calculated according tothe following formula:mg BSA bound per ml suction drained adsorbent=(1−(OD of testsupernatant/OD of BSA starting solution))×60 mg BSA/ml adsorbent.Washing

In a preferred embodiment the washing liquid is water e.g. tap water,demineralised water, water produced by reverse osmosis or distilledwater.

In a particularly interesting embodiment of the invention, the washingand eluting step is combined into one step, wherein it is one and thesame liquid that is employed for washing out the impurities as well assubsequently eluting the product.

In a preferred embodiment of the present invention the flow rate usedfor the washing steps involved is selected from the ranges outlinedpreviously for applying the protein-containing mixture to the adsorbentcolumn.

Elution

The biomolecular substances of interest are released from the adsorbentmedium by using an elution buffer, which produces a generally clear andconcentrated solution of product.

In order to obtain the purified protein or proteins to be isolated theelution may be performed by any method conventionally described andknown in the prior art.

In an alternative and very suitable embodiment of the present invention,the elution of the adsorbed protein is performed with a solution,typically selected from the group consisting of dilute base, diluteacid, and water. In the embodiment wherein the eluting or washing stepis performed with such a solution, the solution is dilute so as tominimise the amount of salt and other unwanted substances present in theeluted product.

Thus, in a preferred embodiment the dilute acid or base used for elutionof the biomolecular substance has a salt concentration of less than 50mM, preferably less than 30 mM, even more preferable less than 20 mM.The determination of the salt concentration is performed directly on theeluate fraction containing the protein or proteins to be isolatedwithout additional dilution of the eluate fraction. Common, low cost andnon-toxic acids and bases are applicable. Specifically preferred are thebases sodium hydroxide (NaOH), potassium hydroxide (KOH), calciumhydroxide (Ca(OH)₂), ammonium hydroxide (NH₄OH).

In a preferred embodiment of the present invention the flow rate usedfor the elution step or steps involved is selected from the rangesoutlined previously for applying the protein-containing mixture to theadsorbent column.

The Product

In some instances, more than one molecule of interest is adsorbed to theadsorbent, and sequential elution may be performed in order to increasethe number of products, i.e. one protein is released from the adsorbentin a way that the second protein remains adsorbed. Subsequently, thesecond protein is released under altered conditions, such as analternative eluent.

In the present context, the term “protein” and the term “biomolecularsubstance” are used interchangeable and relates to a compound ofbiological origin comprising at least two amino acids, such as peptide,polypeptide, lipoprotein, lipopolypeptide, glycopeptide, glycoprotein,enzyme, antibody and immunoglubulin.

In a preferred embodiment of the present invention the protein isolatedfrom the protein-containing mixture is selected form the groupconsisting of lactoperoxidase, lactoferrin, bovine serum albumin,β-lactoglobulin, immunoglobulin, α-lactalbumin and glycomacropeptid.

The process of the invention allows for eluates with high proteinpurity. At least one isolated protein fractionate comprises 70% w/w ofthe total protein, more preferably at least 80% of the total protein andmost preferably at least 90% w/w of the total protein.

In a preferred embodiment of the present invention the purity of theprotein to be isolated measured in the eluate is at least 65%, such asat least 75%, e.g. at least 85%, such as at least 90%, e.g. at least 95%such as at least 98%, e.g. 100%.

The eluate fraction containing the protein product needs to be furtherprocessed in order to obtain a powdered product. A small ultrafiltrationunit concentrates the product from a dilute protein solution to aprotein concentrate prior to drying. The choice of drying techniquedepends on the heat sensitivity of the specific protein.

Further Embodiments of the Present Invention

A suitable embodiment of the EBA process for the isolation andfractionation of biomolecular substances from milk and whey according tothe invention may be summarised by the FIG. 3 and the steps:equilibration (optional), application of milk derived raw material, wash(optional), and elution. Before starting the next process cycle, theadsorbent may be equilibrated again and/or regenerated at a certainfrequency. During the entire process, the adsorbent is expanded by anupwards flow of liquid in the column i.e. the flow direction is notreversed during a process cycle.

In a further preferred embodiment the adsorbent comprise more than onepopulation of particles, each population of particles having differentdensities, i.e. the adsorbent may be a mixture of two particlepreparations having different particle size distributions and differentdensities. Preferably, in an adsorbent mixture of two separatepopulations the population of particles having the lowest mean particlesize will have a higher density than the population of particles havingthe highest mean particle size. As a non-limiting example an adsorbentpreparation may consist of a mixture of two populations of particles Aand B, wherein particle population A has a mean particle size of 100 μmand a density of 2.5 g/ml, while particle population B has a meanparticle size of 50 μm and a density of 4 g/ml. Such a mixed populationof adsorbent particles have been shown to exhibit surprisingly highdynamic binding capacity (i.e. high binding efficiency) at flow ratesabove 10 cm/min (i.e. high productivity), while still being expanded toa degree below H/H0=5.0 or even below H/H0=3.0.

It is a further object of this invention to provide a process forisolation and fractionation of biomolecular substances from milk, skimmilk, whey and other milk derived raw materials based on adsorption toany type of solid phase material of any shape and format includingpacked bed adsorption, batch adsorption, suspended bed adsorption, EBAand membrane based adsorption characterised by the use of specificprocess conditions enabling the elution of the bound substances from theadsorbent such that the final salt concentration in the eluate is keptbelow 50 mM salt.

Adsorptive processes are basically generally disclosed by the use of oneadsorption step resulting in one or more isolated products. Although thetechnological possibility to use multiple consecutive adsorption stepson e.g. human serum is well known no such modular process flow sheetshave been suggested or realised for the industrial isolation andfractionation of biomolecular substances from milk or milk derived rawmaterials. However, such a modular process concept is not yet known inthe milk industry and the method of the invention is particularlyamenable and highly attractive to the milk industry since there is aneed to uncouple the production of one product from other products inorder to obtain maximal flexibility in the production set up and therebyto be able to address fluctuating market needs and the possibility tomake use of the non-bound fraction in a more flexible way (e.g. for theproduction of WPC or WPI products).

It is a further object of the present invention to provide a modularprocess for isolation and fractionation of biomolecular substances frommilk, skim milk, whey and other milk derived raw materials based onadsorption to any type of solid phase material of any shape and formatincluding packed bed adsorption, batch adsorption, suspended bedadsorption, EBA and membrane based adsorption characterised by the useof two or more consecutive but independently operated adsorption stepsproviding two or more purified biomolecular substances in amountsaccording to the individual and actual need for each substance.

A further aspect of the invention relates to a process featuring amodular process concept for optimal market adaptation and extension ofthe fractionation scheme.

A suitable embodiment of the modular process of the present invention isexemplified by FIG. 2, relating for illustrative purposes to a wheyprotein fractionation facility: Lactoferrin and lactoperoxidase may beadsorbed to Column I. The run-through from Step I contains the remainingwhey proteins, and Step II captures the immunoglobulin fraction from therun-through. Thus, the run-through from Step II contains the majorportion of the whey proteins that can be further utilised in a WPC orWPI production. The pH-dependent binding chemistry in Step II alsoenables the extraction of bovine albumin and β-lactoglobulin.

In each step, the number and size of columns connected in parallel maybe adjusted in order to obtain optimal market adaptation. Also,extension of a fractionation facility is possible so that new proteinsof interest can be isolated. This is accomplished by adding anothercolumn set with the appropriate ligand chemistry for further selectiveprotein extraction from the run through fraction.

In the modular process, at least one adsorbent comprises a polymericmaterial.

It is a particular object of this invention to provide a modular processfor the production of purified lactoperoxidase, lactoferrin,β-lactoglobulin, bovine albumin, immunglobulin G, α-lactalbumin andglycomacropeptide from milk, skim milk, whey and other milk derived rawmaterials, which process comprises at least two consecutive adsorptionsteps wherein the first adsorption step provides purifiedlactoperoxidase and lactoferrin and the second adsorption step providesbeta-lactoglobulin, albumin and immunoglobulin G in one or morefractions.

The modular process comprises two or more modular units typicallyconnected in series.

It is a further object of this invention to provide a process for theproduction of a mixture of immunoglobulin G, lactoferrin andlactoperoxidase in one adsorption step.

In an embodiment of the present invention the mixture comprises at least2 proteins and said fractionation of the at least 2 proteins isperformed using an expanded adsorbent bed or packed adsorbent bed orcombinations thereof.

The fractionation is carried out with at least 2 adsorbents each placedin a modular unit.

The steps for fractionation of the proteins from the mixture comprisesthe steps of:

-   i) optionally adjusting the pH in the mixture;-   ii) optionally equilibrating the adsorbent;-   ii) applying the solution to the adsorbents;-   iii) optionally washing with a liquid;-   iv) eluting the adsorbed proteins with one or more eluents selected    from the group consisting of dilute acid, dilute base and water;-   vi) isolating at least one protein mixture comprising a single    protein or various proteins.

In a further embodiment of the present invention, the modular processinvolves the in-line isolation of at least one of the proteins selectedfrom the group consisting of lactoperoxidase, lactoferrin, bovinealbumin, β-lactoglobulin, immunoglobulin α-lactalbumin andglycomacropeptide or mixtures thereof from a solution wherein saidsolution comprising at least one of the above mentioned proteins, saidmethod comprises the steps of:

-   i) optionally adjusting the pH of the solution;-   ii) applying said solution to an optionally equilibrated adsorption    column comprising an adsorbent, said adsorbent comprises a particle    with at least one high density non-porous core, surrounded by a    porous material and the adsorbent has a particle density of at least    1.5 g/mL and mean particle size of at most 150 μm;-   iii) optionally washing with a liquid:-   iv) eluting at least one protein from each modular unit using one or    more aqueous eluents and wherein the final concentration of salt in    the eluate is kept below 50 mM.

In a preferred embodiment of the present invention a plug flow in theadsorbent layer present in the column is provided. This plug flow isestablished by supplying a layer of inert glass beads to the column andpositioned in the stirred zone or in the area of the inlet of thecolumn.

It is a further object of this invention to provide uses for isolatedprotein products produced by any of the invented production processes.

The present investigators have found that the preferred method forintroducing intractable raw materials into a column is throughrelatively large openings. The fluid distribution in FastLine columns isbased on a rotating sprinkler principle. An inlet valve for the rawmaterial is located below the base of the column and the raw materialenters the column through large diameter holes (3 mm) in the hollow armsof the sprinkler. When the process is running, the sprinkler gentlyrotates, creating a sweeping motion along the entire cross-section ofthe column. Due to the gentle stirring action of the sprinkler, themixed zone in the expanded bed is limited to a narrow zone located atthe bottom of the column, and plug-flow behaviour without back-mixing isobtained in the upper part of the column.

The column may be the UpFront's FastLine® column for EBA which isdisclosed in co-pending application no. PCT/DK01/00332.

Other types of fluid bed columns have been described in WO 92/00799, WO92/18237 and WO 99/65586.

The following examples and drawings will illustrate the inventionfurther.

EXAMPLES Example 1

Characteristics of an EBA Adsorbent.

This example describe the features of the adsorbent from UpFrontChromatography used in Example 2-19.

The term “UpFront Stainless Steel-Agarose” refers to adsorbent particlesproduced by an emulsification process as conceptually described in thedescription. They comprise a core material consisting of stainless steelbeads (1-PSR 2, Anval, Sweden) sized in the range 22-44 μm. A layer ofagarose (⁴% agarose) surrounds the stainless steel core bead(s). Theadsorbent particle population is sieved to get a particle sizedistribution in the range of 30-120 μm (see FIG. 4). The mean particlediameter has been determined to be 65 μm by a microscopic examinationcombined with image processing.

The particle population of the adsorbent comprises both compositeparticles having a pellicular structure (i.e. one steel bead embedded inthe agarose polymeric phase) as well as conglomerate particles havingmore than one steel bead inside the agarose phase The density of theadsorbent particles was estimated to be 2.3 g/ml by the followingmethod:

A sample of the “UpFront Stainless Steel-Agarose” adsorbent was washedcarefully on a suction filter and hereafter drained for the interstitialwater by gentle suction in 5 minutes. Hereafter 30,0 gram of the wet butsuction drained adsorbent was weighed into a measuring cylinder followedby the addition of 50.0 ml water. After thorough mixing of the adsorbentparticles with the added water the total volume (Vt) of the resultingsuspension was read on the measuring cylinder. The density of theadsorbent particles was then calculated according to the formula:d=30/(Vt−50)g/ml.

The volume fraction of the adsorbent particles that constitutes theagarose phase can be estimated by assuming an approximate density of thestainless steel beads to be 8.0 g/ml and the density of the agarosephase to be 1.0 g/ml. On these assumptions the volume fraction of theadsorbent particles constituted by the agarose phase will be approx. 81%v/v.

Example 2

Epoxy Activation and Cross-Linking of “UpFront Stainless Steel-Agarose”with a Poly-Epoxy Compound.

The adsorbent “UpFront Stainless Steel-Agarose” described in example 1was chemically activated and cross-linked by the use of a polymericepoxy compound “GE 100”, HS code 2910 90 00, CAS-no.:90529-77-4/25038-04-4 from RASCHIG GmbH, Ludwigshafen, Germany followingthe procedure below:

25 L of wet but suction drained “UpFront Stainless Steel-Agarose” wasmixed with 20 L demineralised water, 10 kg sodium sulphate (water free),2.5 L 32.5% (w/w) sodium hydroxide and 1.25 L “GE 100”. The suspensionwas thoroughly mixed at room temperature for 1 hour. At this timeanother 1.25 L “GE 100” was added to the stirred suspension, followed bymixing at room temperature for another hour. The addition of 1.25 L “GE100” followed by mixing for one hour was repeated for another threetimes, so that the suspension totally had been added 5×1.25 L “GE 100”.

Following reaction with “GE 100” the adsorbent was washed with 250 L,hot (40° C.) water on a suction filter and 250 L water having roomtemperature.

Example 3

Preparation of a Sulfonic Acid Ion Exchanger with Butane Sultone.

The “GE-100” activated and cross-linked adsorbent prepared in Example 2was further reacted with butane sultone to prepare a cation exchangercomprising sulfonic acid groups.

25 L “GE 100” reacted “UpFront Stainless Steel-Agarose” from Example 2was washed on a suction filter with 50 L 32.5% w/w sodium hydroxide,followed by draining by gentle suction. The highly basic adsorbent cakewas transferred into a jacketed and termostated reaction tank and added15 L deionised water. The adsorbent was suspended in the aqueous phaseby gentle stirring and the suspension was then heated to 70° C.

The suspension was then added 1 kg sodium dodecyl sulfate, which wasdissolved under gentle stirring. While continuing stirring andmaintaining the temperature close to 70° C. 1,4-butane sultone, code13671, CAS No.: 1633-83-6, ORGANICA, Wolfen, Germany, was added in 8portions each of 1.56 L butane sultone. In between each addition of thebutane sultone the suspension was allowed to react under stirring for 20min. Following the last addition of butane sultone the adsorbentsuspension was stirred for another 20 min.

After reaction with butane sultone the adsorbent was again washed on asuction filter with 500 L water followed by washing in an expanded bedcolumn (Ø=30 cm) with another 500 L water using a linear flow rate of 15cm/min. The washed adsorbent now comprising cation exchanging sulfonicacid groups was then drained and stored in 20% ethanol. The contents ofsulfonic acid groups covalently coupled to the agarose phase of theadsorbent was determined by acid-base titration to be 125 millimole perliter wet but drained adsorbent.

Example 4

Epichlorohydrin Activation of “UpFront Stainless Steel-Agarose”

30 L “UpFront Stainless Steel-Agarose” described in example 1 was washedwith demineralised water and drained on a suction filter. The adsorbentcake was then transferred into a jacketed and termostated reaction tankand added 24 L deionised water. The adsorbent was suspended in theaqueous phase by gentle stirring and the suspension was then heated to40° C. Following this 3 L 32.5% w/w sodium hydroxide and 3.8 Lepichlorohydrin was added whereafter the suspension was stirred at 40°C. for 2 hours.

Following the reaction with epichlorohydrin the adsorbent was washedwith 500 L deionised water at room temperature.

The content of epoxy groups was determined by titration with thiosulfateto correspond to 35 millimole per liter wet but drained adsorbent.

The epoxy activated adsorbent was then further reacted with a ligand asdescribed in example 5.

Example 5

Coupling of 2-Mercapto-Benzoic Acid to Epoxy Activated Adsorbent.

The epichlorohydrin activated adsorbent produced according to example 4was further reacted with the ligand 2-mercapto-benzoic acid by thefollowing procedure. One liter of suction drained epoxy activatedadsorbent was added 1 liter of a solution of 100 g/L 2-mercapto-benzoicacid titrated to pH 11 with 5 M sodium hydroxide. The adsorbentsuspension was then mixed gently for 18 hours at room temperature,followed by washing with 20 liters of deionised water.

The amount of 2-mercapto-benzoic acid covalently coupled to theadsorbent was determined by acid-based titration to be 31 millimoles perliter wet but drained adsorbent.

Example 6

Coupling of 2-Mercapto-Nicotinic Acid.

The epichlorohydrin activated adsorbent produced according to Example 4was further reacted with the ligand 2-mercapto-nicotinic acid byfollowing the procedure described in Example 5.

The ligand concentration was determined to be 32 millimoles per literdrained adsorbent.

Example 7

EBA on Sweet Whey for Isolation and Purification of Lactoperoxidase andLactoferrin

An EBA column (diameter=2 cm), FastLine 20, cat. no.7020-0000, UpFrontChromatography A/S, Denmark, was placed on a magnetic stirrer and 15 mLdemineralised water was added into the column. Solid glass beads 150-250μm (K. Hoyer Christensen, Denmark) were added into the column until apacked bed height of 10 cm was reached followed by adding 10 cm packedbed height (31 ml) of the sulfonic acid cation exchanger prepared inexample 3. The solid glass beads were added to function as a layer ofinert beads positioned in the stirred zone at the bottom of the columnthroughout the process.

This may generally result in a more efficient exploitation of theadsorbent since a higher fraction of the adsorbent then will bepositioned above the stirred zone (in the plug flow zone with minimalback-mixing) as compared to a setup without added glass beads at thebottom of the column.

The column was connected to a UV monitor (Amersham Pharmacia Biotec AB,Sweden, optical unit UV-1) and a recorder (Pharmacia Sweden,Pharmacia/LKB Rec.1). The magnetic stirrer (Janke & Kuinhel BMBH & Co.,IKA MAG REO) was started and the column was washed for 15 minutes withtap water using a linear flow of 10 cm/min. A fresh sample ofnon-pasteurised bovine sweet whey (from the production of Mozarellacheese) was adjusted to pH 6.5 with 1 M NaOH (Merck cat. no. 1.06498)and 6.2 L whey was then loaded onto the column (adsorbent:whey ratio1:200) using a linear flow of 7.5 cm/minute.

When all the whey was loaded the column was washed with tap water(conductivity 0.65 ms/cm) until the UV-baseline was reached on therecorder (2.0 AUFS). The degree of expansion of the adsorbent (H/H0,i.e. the expanded bed height at a given flow rate in relation to thepacked bed height at zero flow) was recorded visually during theexperiment.

Lactoperoxidase (LP), which had been bound to the adsorbent was theneluted with 25 mM K₂HPO₄ (Merck cat. no.1.05101), 0.3 M NaCl (Baker,cat.no.0319) pH adjusted to 6.5 with HCl ( Merck cat no. 1.00316). Theeluted LP peak was collected according to the recorded UV-signal.Lactoferrin (LF), which also was bound to the adsorbent together with LPwas eluted with 20 mM NaOH (Merck cat. no. 1.06498) and the LP peak wascollected according to the recorded UV-signal. Following elution pH inthe LF eluate was adjusted to a pH between 6 and 8 with 1 M HCl (Merckcat 1.00316).

The yield of LP and LF was estimated by measuring the Optical Density,OD 280 nm, of the eluates on a Spectronic, Cecil CE2041,UV-spectrophotometer.

The yield of LP and LF in the eluates was then calculated using thefollowing extinction coefficients for the two proteins:Lactoperoxidase E_(280, 1 mg/ml, 1 cm)=1.2Lactoferrin E_(280, 1 mg/ml, 1 cm)=1.0

Following this the yield of mg LP and mg LF per liter whey loaded couldbe calculated.

The purity of the eluates was determined by SDS Polyacrylamideelectrophoresis using preceast gels: SDS Page 4-20% Tris-Glycine Gel 1.0mm cat. no.345-0033 BioRad, running buffer cat. no.EC2675 Invitrogen andsample buffer cat. no.EC2676, Invitrogen. The eluates and the crude wheyraw material were diluted 1+1 with the non-reducing sample buffer andheated on a water bath at 100° C. for 5 minuets. 20 μl of each samplewas loaded on the gel. The electrophoresis apparatus, BioRad, wasconnected to a power supply and the voltage was set to 200 volt. Thepower was turned off when the blue marker reached the bottom of the gel.The SDS gel was stained a Colloid Blue Staining Kit LC6025, Invitrogenfor 18 hours on a shaking table and destained in water.

Dry matter in the eluates was determined by freeze drying (Hetosicc,Heto Holten Denmark), Before freeze drying, the eluates were dialysedagainst demineralised water at 4° C. in a ratio of 1 vol of eluate to100 volumes of water. 4 changes of water over 2 days were performedbefore the eluate was freeze dried.

Conductivity after ended dialysis was measured (Crison conductimeter525) and 50 ml of the dialysed eluate was freeze dried overnight. Thedry matter was determined by weighing the residual.

Results:

Expansion of matrix (incl. Inert glass beads) at a liner flow at 7.5cm/min Step Expanded matrix H Expansion H/H₀ Load 29 cm 1.45 Wash 27 cm1.35 LP elution 27 cm 1.35 LF elution 27 cm 1.35 Packed bed height H₀ =20 cmYields of LF and LP

Measuring of OD 280 nm: Peak 1 (LP) 0.785

-   -   Peak 2 (LF) 2.080

Volume of eluates: Peak 1 (LP) 250 ml

-   -   Peak 2 (LF) 200 ml        Total Yield of LP and LF

${{LP}\frac{0.785*250{ml}}{1.2}} = {164\mspace{14mu}{mg}\mspace{14mu}{LP}}$${{LF}\frac{2.080*200\mspace{14mu}{ml}}{1}} = {416\mspace{14mu}{mg}\mspace{14mu}{LF}}$Yield of LP and LP per Liter Whey Loaded.

${{LP}\mspace{11mu}\frac{164\mspace{14mu}{mg}}{6\mspace{14mu}{liter}}} = {27\mspace{14mu}{mg}\mspace{14mu}{LP}\text{/}{liter}\mspace{14mu}{whey}\mspace{11mu}{loaded}}$${{{LF}\mspace{14mu}\frac{416\mspace{14mu}{mg}}{6\mspace{14mu}{liter}}} = {69\mspace{14mu}{mg}\mspace{14mu}{LF}\text{/}{liter}\mspace{14mu}{whey}\mspace{14mu}{loaded}}}\text{}$Freeze Drying of the Eluates:

Conductivity after ended dialysis LP eluate 20 μS

-   -   LF eluate 16 μS

Yield determined as dry matter LP 32 mg/L whey loaded

-   -   LF 63 mg/l whey loaded

As can be seen there is a good correspondence between thespectrophotometric determination of the yields and the yields determinedby measuring the dry matter in each eluate.

Purity of Eluted LP and LF

The purity of the eluted proteins was determined by SDS-PAGE. As can beseen from FIG. 5 the eluted LP and LF has been isolated and purifiedvery efficiently.

Lane Purity 1 Whey 2 Wash 3 LP eluate >70% 4 LF eluate >90% EBA on sweetwhey having different pH values during load.

Example 8

EBA on Sweet Whey Having Different ph Values During Load

Three experiments were performed labelled A, B and C. Three FastLine 20columns were loaded with inert glass beads and active sulfonic acid ionexchanger. Each FastLine 20 column was packed with 10 cm inert glassbeads and 5 cm packed bed height of the sulfonic acid cation exchangerprepared in Example 3.

For column A the non-pasteurised sweet whey (from the production ofMozarella cheese) was adjusted to pH 6.5, for column B to pH 7.0 and forcolumn C the whey was adjusted to 7.5 using 1 M NaOH.

The experiments illustrate the effect of varying pH on yield and purityof the isolated lactoperoxidase and lactoferrin. The sweet whey wasloaded on the column at a linear flow rate at 7.5 cm/min in a ratiobetween adsorbent and whey of 1:200. (materials and set up see Example7.

Whey loaded Column at pH A 6.5 B 7.0 C 7.5Results:

Yields from measuring of OD280 nm of eluates:

Yield of LP Yield of per L whey LF per L Column pH loaded whey loaded A6.5 27 mg 65 mg B 7.0 29 mg 71 mg C 7.5 21 mg 73 mg

Determination of purity by SDS PAGE

Purity of Purity of Column pH LP eluate LF eluate A 6.5 80% 95% B 7.070% 90% C 7.5 65% 90%

Example 9

EBA on Sweet Whey. Variation of Flow Rate.

Four experiments were performed labelled A, B, C and D. The experimentsillustrate the effect of varying the flow rate during load of the wheyon the yield and purity of the isolated lactoperoxidase and lactoferrin.

Four columns were packed. Each FastLine 20 column was packed with 10 cmglass beads and 5 cm packed bed height of the sulfonic acid cationexchanger prepared in example 3.

Non-pasteurised sweet whey (from the production of Mozarella cheese)adjusted to pH 6.5 was loaded on the column in a ratio at 1:200 and 4different columns were run with different linear flow. (materials andset up see example 7.

Column Linear flow A 7.5 cm/min  B 10 cm/min C 15 cm/min D 25 cm/minResults:

Yield from measuring of OD280 of eluates.

Yield of LP per L Yield of LF per Column Linear flow whey loaded L wheyloaded A 7.5 cm/min  29 mg 71 mg B 10 cm/min 29 mg 60 mg C 15 cm/min 27mg 61 mg D 25 cm/min 19 mg 55 mg

Determination of purity from SDS Page

Purity of Purity of Column Linear flow LP eluate LF eluate A 7.5 cm/min 75% 90% B 10 cm/min 75% 90% C 15 cm/min 75% 90% D 25 cm/min 75% 90%

Example 10

EBA on Sweet Whey. Variation of Adsorbent: Whey Ratio.

Three experiments were performed labelled A, B and C. The experimentsillustrate the effect of varying the ratio between the amount ofadsorbent and the amount of whey loaded on the yield and purity of theisolated lactoperoxidase and lactoferrin.

Three FastLine 20 columns were packed. Each FastLine 20 column waspacked with 10 cm glass beads and 5 cm packed bed height of the sulfonicacid cation exchanger prepared in Example 3.

The non-pasteurised sweet whey (from the production of Mozarella cheese)was adjusted to pH 6.5 and loaded on the column at a linear flow at 7.5cm/min. Column A , B and C were loaded with whey in different ratiobetween matrix and whey (materials and set up see Example 7).

Column Ratio Matrix:whey A 1:200 B 1:300 C 1:400Results:

Yield from measuring of OD280 of eluates.

Yield of LP per Yield of LF per Column Ratio L whey loaded L whey loadedA 1:200 27 mg 65 mg B 1:300 31 mg 52 mg C 1:400 35 mg 40 mg

Determination of purity from SDS Page

Purity of Purity of Column Ratio LP eluate LF eluate A 1:200 80% 95% B1:300 80% 95% C 1:400 80% 95%

Example 11

EBA on Sweet Whey. Variation of Loading Temperature.

Three experiments were performed labelled A, B and C. The experimentsillustrate the effect of varying the temperature during load of the wheyon the yield and purity of the isolated lactoperoxidase and lactoferrin.

Three FastLine 20 columns were packed. Each FastLine 20 column waspacked with 5 cm packed bed height of the sulfonic acid cation exchangerprepared in example 3 (i.e. no inert glass beads were added in theseexperiments).

The non-pasteurised sweet whey (from the production of Danbo cheese) wasadjusted to pH 6.5 and loaded on column A at +4° C., on column B at 22°C. and at column C at 50° C. The whey was loaded with a linear flow rateof 7.5 cm/min and in a ratio between matrix and whey of 1:300. Expansionof the matrix was measured during the run (materials and set up seeExample 7).

Loading Column temperature A  +4° C. B +22° C. C +50° C.Results:

Yield from measuring of OD280 nm of eluates

Yield of LP Yield of LF Loading per L whey per L whey Column temperatureloaded loaded A  +4° C. 14 mg 15 mg B +22° C. 12 mg 30 mg C +50° C. 19mg 19 mg

Determination of purity from SDS Page

Loading Purity of Purity of Column temperature LP eluate LF eluate A +4° C. 60% 90% B +22° C. 60% 90% C +50° C. 70% 90%

Determination of expansion during the run

Expanded to (H) Expansion H/H₀ Column A Load of whey 22 cm 4.4 Wash 16cm 3.2 Elution 14 cm 2.8 Column B Load of whey 14 cm 2.8 Wash 10 cm 2Elution 10 cm 2 Column C Load of whey 12 cm 2.4 Wash 13 cm 2.6 Elution14 cm 2.8 Settled bed height H₀ = 5 cm

Example 12

EBA on Skim Milk.

This experiment illustrates the use of EBA according to the inventionusing skim milk as raw material.

A FastLine 20 column was packed with 10 cm inert glass beads and 5 cmpacked bed height of the sulfonic acid cation exchanger prepared inExample 3. Skim milk (at its natural pH of 6.7) was loaded on the columnat a linear flow at 10 cm/min and in a ratio between matrix and skimmilk of 1:200. Expansion of the adsorbent was measured during the run(materials and set up see Example 7).

Results:

Yield from measuring of OD280 nm of the eluates.

Yield of LP per L whey Yield of LF per loaded L whey loaded 54 mg 125 mg

Determination of purity from SDS Page

Purity of Purity of LP eluate LF eluate 80% 90%

Determination of expansion during the run

Expanded to (H) Expansion H/H₀ Load 28 cm 1.9 Wash 20 cm 1.3 Elution 19cm 1.3 Settled bed height H₀ = 15 cm

Example 13

Packed Bed Isolation of Immunoglobulins from Whey: Binding of IgG to2-Mercaptobenzoic Acid Resin Depending on the pH of the Whey Loaded ontothe Adsorbent.

Bovine IgG can be isolated from acid or sweet whey by the use of theadsorbent coupled with the ligand 2-mercaptobenzoic acid described inexample 5.

Five packed bed columns (diameter=0.5 cm) each containing 1 ml ofadsorbent were equilibrated with 5 ml of demineralized water. Acid wheywas adjusted to respectively pH 4.0, 4.5, 5.0, 5.5 and 6.0 and loaded tothe columns. 20 ml whey of a given pH value was loaded onto each column.

The non-bound proteins were washed out with respectively 10 mM sodiumcitrate pH 4.0, 4.5, 5.0, 5.5 and 6.0 corresponding to the pH of the rawmaterial used for that particular column. 7 ml washing buffer was usedfor each column.

All columns were eluted with 5 ml of 20 mM tris pH 9.5 followed by 5 mlof 20 mM potassium phosphate, 1 M sodium chloride pH 12.0.

The flow rate used during the entire process was 300 cm/hr.

The eluates were analyzed by SDS-PAGE to determine the qualitativecomposition of the bound proteins. SDS-PAGE: 4-20% gradient gel fromNovex (USA). Non-reduced and Coomassie stained.

Results:

The SDS-PAGE as shown in FIG. 6 illustrates the content of proteins inthe two eluates obtained from column.

The SDS-PAGEs show that when the pH is increased in the whey loaded ontothe column the binding capacity of IgG is decreased. The purity of theIgG increases when the pH in the loaded whey is increased. The optimumbinding capacity of IgG is approx. pH 5.0.

Example 14

EBA. Isolation of Immunoglobulins from Whey Already Absorbed forLactoferrin and Lactoperoxidase.

A 2 cm diameter EBA column, FastLine 20, from UpFront Chromatography A/S(Cat. no.: 7020-0000) was loaded with 10 cm packed bed height of inertglass beads and 15 cm (47.1 ml) of the adsorbent coupled with the ligand2-mercaptobenzoic acid described in example 5. The glass beads (150-250μm, density 2.5 g/ml) are used as a filler in the bottom of the columnwhere the stirrer is rotating. The mixed zone created by the magnetoccurs in the glass bead layer resulting in a perfect plug flow throughthe active adsorbent.

The column was equilibrated with 236 ml of demineralized water. Sweetwhey depleted for lactoferrin and lactoperoxidase i.e. run through froma cation exchanger column procedure as described in example 7 waspH-adjusted to pH 4.9 with 1 M HCl. The column was loaded with 942 ml ofthe pH adjusted whey i.e. an adsorbent: whey ratio of 1:20. The flowrate during the whole process was 450 cm/hr=1.4 l/hr.

After loading of the whey non-bound proteins were washed out with 530 mldemineralized water.

After washing out non-bound proteins with demineralized water, thecolumn was washed with 2.5 mg/ml caprylic acid pH 6.0. Volume of washwith 2.5 mg/ml caprylic acid pH 6.0: 471 mL.

The bound IgG was then eluted with 20 mM NaOH. The raw material, thewashing fraction with caprylic acid and the eluate was analysed by SDSPAGE. SDS-PAGE: 4-20% gradient gel from Novex (USA). Non-reduced andCoomassie stained. 25 μl sample in each well.

Results:

The SDS-PAGE shown in FIG. 7 illustrates the content of proteins in theraw material, wash fraction with caprylic acid and in the eluate.

As can be seen from the figure the wash fraction with caprylic acidconstitutes a purified solution of beta-lactoglobulin (β-LG) and bovinealbumin (BSA). No alfa-lactalbumin is present in this fraction. It canfurther be seen that the eluate fraction constitutes a highly purifiedIgG containing only minor impurities of other milk protein components(estimated purity >85%).

Example 15

EBA Isolation of Immunoglobulins from Whey and Recovery of Pureβ-Lactoglobulin:

As illustrated in Example 14 the adsorbent coupled with the ligand2-mercaptobenzoic acid produced as described in example 5 also binds BSAand beta-lactoglobulin (β-LG) at pH 4.9. This experiment illustratesthat with certain washing buffers it is possible to obtain a fraction ofpure β-LG, a fraction comprising both β-LG and BSA and an elutionfraction containing mainly IgG.

An experiment was carried out according to example 14 with the followingmodifications: After washing out non-bound proteins with demineralizedwater the column was further washed with 50 mM sodium acetate pH 5.5.Volume of wash with sodium acetate: 471 ml. The column was then furtherwashed with 2.5 mg/ml caprylic acid pH 6.0.

Volume of wash with caprylic acid: 330 ml.

The IgG was finally eluted with 20 mM NaOH.

All fractions were analyzed by SDS PAGE. SDS-PAGE: 4-20% gradient gelfrom Novex (USA). Non-reduced and coomassie stained. 25 μl sample ineach well.

Results:

The SDS-PAGE shown in FIG. 8 illustrates the content of proteins in thetwo washes and the eluate.

The SDS-PAGE shows that washing with 50 mM sodium acetate pH 5.5 resultsin a fraction containing pure β-LG (lane #1). Wash with 2.5 mg/mlcaprylic acid pH 6.0 results in a fraction containing BSA and β-LG (lane#2). The eluate (235 ml) contains a highly purified IgG (estimatedpurity >65%)

Example 16

EBA Isolation of Immunoglobulins from Whey: Capacity and Yield of atDifferent Adsorbent: Whey Ratios.

The adsorbent: whey ratio (liters of whey loaded per liter adsorbent)was varied to find the capacity of the matrix-and optimal yield of IgGper liter whey.

Experiment was carried out according to example 14 with the followingmodificaiotns.

In three consequtive-experiments the EBA column was loaded with 707 ml,942 ml and 1,413 ml respectively of sweet whey (from the production ofMozarella cheese) adjusted to pH 4.9 resulting in adsorbent:whey ratios(v/v) of 1:15, 1:20 and 1:30 respectively. After load the columns werewashed with 2.5 mg/ml caprylic acid pH 6.0.

Volume of wash for all three columns: 530 ml. Flow rate for all threecolumns: 450 cm/hr=1.4 l/hr.

The IgG was eluted by 20 mM NaOH.

Results:

The table below shows the capacity and yield obtained with the2-mercaptobenzoic acid adsorbent at different whey ratios.

The amount of IgG was calculated from spectrophotometrical measurementson the eluates at 280 nm using an extinction coefficient of E^(1%)_(280 nm)=13. The volumes of the eluates were respectively 170 ml, 174and 226 ml.

Capacity, Yield, Whey ratio, mg IgG in mg IgG/ml mg IgG/l l/l resineluate resin whey 15 331 7 468 20 395 8.4 419 30 578 12.3 409

The above table shows that the highest yield per liter whey is obtainedat a whey ratio of 1:15 resulting in 468 mg IgG/mi whey.

The capacity of the resin increases by 75% when the whey ratio isincreased from 15 to 30 liter whey loaded per liter resin.

Example 17

EBA Isolation of Immunoglobulins from Whey: Capacity and Yield as aFunction of Flow Rates During Load of Whey:

Experiments were carried out as described in example 14 with thefollowing modifications. In three consequtive experiments the flow ratesused during loading of the raw material (sweet whey, from the productionof Mozarella cheese adjusted to pH 4.9) were respectively 300, 450 and600 cm/hr.

After loading, the columns were washed with 2.5 mg/ml caprylic acid pH6.0.

Volume of wash for all three columns: 530 ml. Flow rate during wash forall three columns: 450 cm/hr=1.4 l/hr.

The IgG was then eluted with 20 mM NaOH using a flow rate 450 cm/hr=1.4l/hr for all three columns.

Results:

Expansion during load of the three columns was respectively 1.7 (300cm/hr), 2 (450 cm/hr) and 2.3 (600 cm/hr).

The table below shows the capacity and yield obtained with the2-mercaptobenzoic acid adsorbent at different flow rates used duringload of the whey.

The amount of IgG was calculated from spectrophotometrical measurementsat 280 nm. Volumes of the eluates were respectively 212 ml, 175 ml, and156 ml.

Flow rate, during Capacity, Yield, whey load, mg IgG in mg IgG/ml mgIgG/l cm/hour eluate resin whey 300 430 9.1 457 450 395 8.4 419 600 2936.2 311

The table shows that the highest yield per liter whey applied isobtained at a flow rate of 300 cm/hr during load of 457 mg IgG/ml whey.

The capacity of the resin is decreased by approximately 30% from 9.1 to6.2 mg IgG/ml resin when the flow rate is increased from 300 to 600cm/hr.

Example 18

Integrated Modular EBA Process for Purification of LP, LF and IgG fromWhey:

This Example illustrates that it is possible to isolate LP, LF and IgGwith the two step process design shown in FIG. 2, where the pH of theeffluent from the first step is adjusted to pH 4.9 before entering thesecond step.

The 2 columns used for this integrated process were 30 cm EBA columns(FastLine 300) from UpFront Chromatography A/S (cat. no.: 7300-0000).

Column No. 1: Purification of LP and LF

Bed height: 10 cm of inert glass particles, 15 cm of the sulfonic acidcation exchanger (10.6 l) described in Example 3.

The experiment was otherwise carried out according to example 7 with thefollowing modifications.

Volume of equilibration: 53 l.

Whey load: 3180 L=adsorbent:whey ratio 1:300.

Volume of wash with water: 160 l.

Flow rate during load and wash: 900 cm/hr.

Volume of LP eluate: 101 l.

Volume of LF eluate: 55 l.

Flow rate during elution: 450 cm/min.

The effluent from column 1 is in-line pH adjusted to 4.9 before loadedonto column 2.

Column No. 2: Isolation of IgG

Bed height: 10 cm of inert glass particles, 15 cm of 2-mercaptobenzoicacid resin (10.6 l) described in example 5.

The experiment was otherwise carried out according to example 14.

Volume of equilibration: 30 l

Whey load: 212 L=ratio 1:20.

Volume of wash with water: 160 l.

Volume of wash with 2.5 mg/ml caprylic acid: 120 l.

Volume of IgG eluate: 40 l.

Flow rate during the whole process: 450 cm/hr.

Results:

The table below shows the yield and purity of the three proteinsrecovered from the two step process design.

The amounts of LP, LF and IgG in each eluate were calculated fromspectrophotometrical measurements on the eluates at 280 nm using E^(1%)_(280, LP)=12. E^(1%) _(280,LF)=10 E^(1%) _(280,IgG)=13

g protein in Yield, mg Protein eluate protein/l whey Purity, % LP 92 31 >90% LF 235 80  >90% IgG 89 419 80-85%

Example 19

Production Estimates.

The following are calculations and production estimates based on theresults obtained during the work with the described adsorbents.

A fractionation facility as described in Example 18 with two large scalecolumns in Step I (Ø=1.5 meter), each containing 265 liters ofadsorbent, can extract lactoferrin and lactoperoxidase from a 690,000liter whey stream per day (24 hours). The approximate productivity is 21kg lactoperoxidase and 52 kg lactoferrin per day corresponding to anextraction yield of 30 mg lactoperoxidase and 75 mg lactoferrin perliter whey. Both products feature high purity (>90% purity).

Immunoglobulin is present in whey in a significantly higherconcentration (0.5-1.0 g/l whey) than lactoferrin and lactoperoxidase.Thus, more columns are needed in Step II in order to extractimmunoglobulin from the whey. A 690,000 liter whey stream per dayoccupies three columns (Ø=1.5 meter) each containing 265 liters ofadsorbent media. The productivity is approximately 277 kg immunoglobulinper day, corresponding to an extraction yield of 400 mg immunoglobulinper liter whey.

Variations in productivity according to whey type and pre-treatment ofthe whey may occur. All the productivity estimates stated here are basedon results from pilot scale trials.

Example 20

EBA Adsorbents Comprising Tungsten Carbide Particles of 8 MicronParticle Size.

The term “UpFront tungsten carbide-8-agarose” refers to adsorbentparticles produced by an emulsification process (tungsten carbideparticles/melted agarose suspension emulsified in hot Vaseline oilfollowed by cooling, washing of the resulting agarose/tungsten carbidebeads and sieving). They comprise a core material of tungsten carbideparticles (WC 8.0, Kennametal Hertel, Germany) sized at 8 μm and adensity of 15.6 g/ml. The adsorbent particles comprise the tungstencarbide particles as a high density core evenly distributed in thespherical agarose bead (see FIG. 9). For comparative purposes threedifferent preparations were analysed with respect to expansion as afunction of flow rate in an EBA column.

FIG. 10 shows the expansion curve on the three preparations: A, B and C

Preparation A was determined to have a density of 2.4 g/ml and aparticle size distribution as illustrated in FIG. 11.

Preparation B was determined to have a density of 3.3 g/ml and aparticle size distribution as illustrated in FIG. 12.

Preparation C was determined to have a density of 3.2 g/ml and aparticle size distribution as illustrated in FIG. 13.

The densities measured for the three preparations can be used forcalculation of the average volume occupied by the tungsten carbideparticles relative to the volume of the whole bead as described in thedescription:

Volume of tungsten carbide in:

-   Preparation A: approximately 10%-   Preparation B: approximately 16%-   Preparation C: approximately 15.4%

The mean particle size can be determined from the particle sizedistribution analysis:

Mean particle size:

-   Preparation A: 56 μm-   Preparation B: 59 μm-   Preparation C: 90 μm

Example 21

EBA Adsorbents Comprising Tungsten Carbide Particles of 25 MicronParticle Size.

The term “UpFront tungsten carbide-25-agarose” refers to adsorbentparticles produced by an emulsification process (tungsten carbideparticles/melted agarose suspension emulsified in hot Vaseline oilfollowed by cooling, washing of the resulting agarose/tungsten carbidebeads and sieving). They comprise a core material of tungsten carbideparticles (WC 25.0, Kennametal Hertel, Germany) sized at 25 μm and adensity of 15.6 g/ml. The adsorbent particles comprise the tungstencarbide particles as a high-density core of one or more tungsten carbideparticles embedded in the spherical agarose bead (see FIG. 14). Onepreparation was analysed with respect to expansion as a function of flowrate in an EBA column.

FIG. 15 shows the expansion curve on the preparation: D

Preparation D was determined to have a density of 3.7 g/ml and aparticle size distribution as illustrated in FIG. 16.

The density measured for the preparation can be used for calculation ofthe average volume occupied by the tungsten carbide particles relativeto the volume of the whole bead as described in the description:

Volume of tungsten carbide in:

-   Preparation D: Approximately 18.5%

The mean particle size can be determined from the particle sizedistribution analysis:

Mean particle size:

-   Preparation D: 77 μm

Example 22

EBA on Sweet Whey. Binding Capacity at High Flow Rate.

The four tungsten carbide-agarose preparations (A-D) described andcharacterised in example 20 and 21 where chemically derivatised asdescribed in example 2 and 3 to produce four preparations of sulfonicacid ion exchangers. These four ion exchangers where then analysed fortheir ability to bind lactoferrin as basically described in example 7.

Four experiments were performed labelled I, II, III and IV. Theexperiments illustrate the lactoferrin binding efficiency of the fourparticle preparations at a flow rate of 25 cm/min during load of thewhey.

Four columns were packed. Each FastLine 20 column was packed with 10 cmglass beads and 5 cm packed bed height of the respective tungstencarbide-agarose-sulfonic acid cation exchanger. The four experimentscomprised the four particle preparations described in example 1 and 2(dette dokument):

-   Column I: Particle preparation A, mean particle size: 56 micron,    density=2.4 g/ml-   Column II: Particle preparation B, mean particle size: 59 micron,    density=3.3 g/ml-   Column III: Particle preparation C, mean particle size: 90 micron,    density 3.2 g/ml-   Column IV: Particle preparation D, mean particle size: 77 micron,    density 3.7 g/ml

Non-pasteurised sweet whey (from the production of Mozarella cheese)adjusted to pH 6.5 was loaded on the columns in a ratio at 1:300 and the4 different columns were run at a linear flow rate of 25 cm/min.

Results:

Yield from measuring of OD280 of eluates.

Yield of LF per Column Linear flow l whey loaded I 25 cm/min 70 mg II 25cm/min 70 mg III 25 cm/min 66 mg IV 25 cm/min 69 mg

Determination of purity from SDS page

Purity of Column Linear flow LF eluate I 25 cm/min 90% II 25 cm/min 90%III 25 cm/min 90% IV 25 cm/min 90%

1. A method for the fractionation of a protein-containing mixturewherein the protein-containing mixture is selected from the groupconsisting of milk, milk derived products, milk derived raw materials,fruit derived products, fruit derived extracts, fish derived products,and fish derived extracts, said method comprising the steps of: a)applying said protein-containing mixture at a flow-rate of at least 5cm/min to an adsorption column comprising an adsorbent, the adsorbenthaving a particle density of at least 1.8 g/ml and a mean particle sizeof at most 150 μm; b) eluting at least one protein from the adsorbent.2. A method according to claim 1, wherein the applying of saidprotein-containing mixture is performed at a flow-rate of about 5-50cm/min.
 3. A method according to claim 1, wherein the adsorbentcomprises a high density non-porous core having a density of at least 4g/ml.
 4. A method according to claim 1, wherein the adsorbent comprisesa high density non-porous core having a density in the range of about4-25 g/ml.
 5. A method according to claim 1, wherein the adsorbent has amean particle size of at most 120 μm.
 6. A method according to claim 1,wherein the adsorbent has a mean particle size in the range of 40 to 150μm.
 7. A method according to claim 1, wherein the adsorbent comprises anon-porous core constituting of at the most 50% of the total volume ofthe adsorbent particle.
 8. A method according to claim 1, wherein theadsorption column is selected from the group consisting of expanded bedadsorption column, a packed bed adsorption column and a combinationthereof.
 9. A method according to claim 1, wherein the adsorbent presentin the column relative to the protein-containing mixture to be loaded onto the column are provided at a ratio of at least 1:1000 measured on avolume/volume basis.
 10. A method according to claim 1, wherein elutingis performed with an eluant selected from the group consisting of dilutebase, dilute acid, and water.
 11. A method according to claim 10,wherein the diluted base is selected from the group comprising sodiumhydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide.12. A method according to claim 1, wherein the elution of the boundsubstances from the adsorbent provides a final salt concentration in theeluate less than 50 mM salt.
 13. A method according to claim 1, whereinthe adsorbent comprises a porous material that comprises a polymericbase matrix.
 14. A method according to the claim 13, wherein thepolymeric base material is a polysaccharide.
 15. A method according toclaim 1, wherein the adsorbent has a binding capacity of at least 10 g/Lof BSA according to test Method A.
 16. A method according to claim 1,wherein at least one protein to be isolated is selected from the groupconsisting of lactoperoxidase, lactoferrin, bovine serum albumin,β-lactoglobulin, immunoglobulin, α-lactalbumin and glycomacropeptide.17. A method according to claim 15, wherein at least one isolatedprotein mixture comprises at least 70% w/w of a single protein.
 18. Amethod according to claim 1, wherein the purity of the protein to beisolated measured in the eluate is at least 65%.
 19. A method accordingto claim 1, wherein the protein-containing mixture comprises at least 2proteins and said fractionation of the at least 2 proteins is performedusing an expanded adsorbent bed or packed adsorbent bed or combinationsthereof.
 20. A method according to claim 1, wherein a plug flow isestablished in the adsorbent by providing a layer of inert glass beadspositioned in the stirring zone.
 21. A method according to claim 1,wherein the particle size is less than 120 μm.
 22. A method according toclaim 1, wherein the particle size is less than 90 μm and the particledensity is at least 1.8 g/ml.
 23. A method according to claim 1, whereinthe particle size is less than 75 μm and the particle density is atleast 2.0 g/ml.
 24. A method according to claim 1, additionallycomprising the step of optionally adjusting the pH of theprotein-containing mixture.
 25. A method according to claim 1,additionally comprising the step of optionally washing the absorptioncolumn.
 26. A method according to claim 1, wherein the absorbentcomprises particles with at least one high density non-porous core,surrounded by a porous material.